Combatting wildflower wounding response

FULL REPORT
This report is taken from the full report Inhibiting the Postharvest Wounding Response in Wild flowers , RIRDC Pub. 
No. 02/114, project number DAV-161A.  It was prepared for RIRDC by Dr Virginia Williamson, Institute of Horticultural Development, Department of Natural Resources and Environment. Phone:03 9210 9222; Fax:03 9800 3521. 
Email:virginia.williamson@nre.vic
Researchers experimenting with a number of wound-inhibiting chemicals and treatments hope to be able to extend the shelf life of Australian wildflowers and increase the range of species suitable for export. 

Their work indicates that an hydrophobic substance, suberin, forms a barrier causing premature wilting because stems are no longer able to take up water from vase solutions. 

It provides practical advice on improving the vase life of a number of Australian wildflowers and recommends easy ways to increase water uptake. 

Thirteen genera/species of Australian cut flowers and foliage were involved in the research, carried out by a team under Dr Virginia Williamson, at the Victorian Department of Natural Resources and Environment’ s Institute of Horticultural Development. Researchers experimenting with a number of wound-inhibiting chemicals and treatments hope to be able to extend the shelf life of Australian wildflowers and increase the range of species suitable for export. 

Their work indicates that an hydrophobic substance, suberin, forms a barrier causing premature wilting because stems are no longer able to take up water from vase solutions. 

It provides practical advice on improving the vase life of a number of Australian wildflowers and recommends easy ways to increase water uptake. 

Thirteen genera/species of Australian cut flowers and foliage were involved in the research, carried out by a team under Dr Virginia Williamson, at the Victorian Department of Natural Resources and Environment’ s Institute of Horticultural Development. 

The Study

The researchers were armed with data showing Australian wildflower exports grew rapidly in the decade to the mid-1990s, then remained fairly stagnant until 1999-2000 when export volume increased to around 5000 tonnes. 

Despite this volume increase in exports, average prices received per kilogram appear to have fallen, earning a total export income of around $31.3 million. The report on the work says while the unit price has been declining steadily for almost 10 years, average prices need to be considered with caution given possible underestimates of flowers sold at auction. 

But the researchers believe that while potential for increased exports has existed for years this has been unfulfilled and increasing vase life could bring improved results. 

Exports currently concentrate on just a few of the 231 Australian native species suitable as cut flowers, mainly kangaroo paw, Banksia, Dryandra and Geraldton Wax. 

To increase the variety of cut flowers exported the researchers realise that flower quality and particularly a good vase life of at least seven days is required. 

This has helped identify the project’ s objectives that include: • counteracting the poor water uptake that characterises most Australian native cut flowers by inhibiting the initial post-harvest wounding response; • determining whether suberin inhibiting treatments can enhance water uptake and increase vase life; • ascertaining by Transmission Electron Microscopy (TEM) whether suberin is deposited in cut stems;and • finding out whether other factors, such as ethylene sensitivity, are involved in a particular flower’ s rapid aging. 

Having selected several species including Acacia , Hakea , Leptospermum , Grevillea , and Crowea , plant material was tested in a specially designed vase life room. Before work started on vase life, the researchers determined pH levels, measured dissolved oxygen, and took bacterial counts. 

They then undertook numerous and different experiments to study hypothesised wounding responses. These included studies of plants using Transmission Electron Microscopy, measurement of water uptake and transpiration, testing of available suberin inhibiting chemicals, washing experiments, and assessment of sensitivity to naturally occurring ethylene. 

The Results

Transmission Electron Microscopy

For the first time TEM was used to examine cut flowers for a wounding response. It showed tangible evidence of the physiological change in stems kept in deionised water for 48 hours compared with those in the suberin inhibitor, Scarvone. 

Changes in the electron density of pit membranes occurred in the stems in the deionised water. But in the S-carvone treated stems, the pit membrane remained intact. 

Although suberin was not observed 48 hours after cutting, the initial stages of pit membrane modification identified may be followed by a deposit of granular/fibrillar material and then suberin. 

Two-stage processes like this can occur, first as an alteration of the pit membranes and secondly as a wound-associated secretion followed a week after the wounding by suberisation of parechyma cells, with secreted material occuring in adjacent xylem vessels and fibres. 

While clear therefore that the formation of suberised structures is still possible it remains questionable whether these early changes in pit membrane structure seen under TEM are enough to inhibit water uptake and reduce vase life. Certainly the stems kept in S-carvone lived significantly longer than the other treatments. 

Recutting stems 

Experiments involving daily recutting of stems were undertaken to determine whether this might remove a suberised layer. Removing one centimetre a day significantly improved the vase lives of Acaia Baileyana , Leptospermum obovatum and Leptospermum polygalifolium which were the species tested. 

Although the researchers speculated on why this increased vase life, when considering the results with those from the TEM analysis, they hypothesised that recutting removed early stages of the wound response. 

This means the existing recommendation to recut stems daily assumes new more meaning ful importance even though each cut will initiate a new wound reaction. But it remains clear that there is need for additional work on this. 

High water verses low water

Experiments showed vase life was significantly improved when high vase water levels were used compared with lower levels. While previously acknowleged the mechanisms have not been understood. 

Tests were undertaken on various hypotheses, including increased water pressure forcing water up the stem and past a blockage, water being taken up through the outside of the stem and bacterial levels being diluted in larger volumes of water. 

These experiments showed it was increased hydrostatic pressure that improved vase life rather than water being taken up through the outside of the stem or increases in bacterial numbers. 

Suberin-inhibiting chemicals

The suberin-inhibiting chemical tests aimed to screen a range of chemicals and concentrations to determine their impact on vase life. 

Acacia Baileyana foliage was chosen for the experiments mainly because it was the readily available flowering material at the time. 

Chemicals included were: • Ascorbic and Citric Acids, chosen for their antioxidant properties; • Abscisic Acid (ABA) , which was known to promote stomatal closure, to induce suberisation and inhibit protein synthesis; • Cycloheximide (CHI) which is known to inhibit suberin synthesis and is a general inhibitor of protein synthesis;and • Salicylhydroxamic Acid (SHAM) because of its known ways of inhibiting suberin formation. 

The antioxidants proved beneficial to vase life. 

ABA tests were abandoned because of complications likely to impact on results. Initial CHI and SHAM experiments also were discarded, the former because concentrations used were considered too high and the latter because of the masking presence of ethanol used to dissolve the SHAM. 

A second experiment using Acacia Baileyana and SHAM dissolved without ethanol resulted in longer vase life, but not significantly different to tests involving water. Lower concentrations of SHAM were considered likely to provide better results. 

Ethylene

The project identified more Australian flowers sensitive to ethylene. The discovery was more a by-product of the work rather than a direct testing of the suberin hypothesis. Species found to be sensitive were:Baeckea virgata, and Crowea exalata. A New Zealand native Lophomyrtus x ralphii’ Krinkly’ foliage also was tested due to the shortage of Australian native plant materials at the time. 

Several additional Australian species require further testing, having shown leaf detachment during the vase life experiments which indicates an ethylene sensitivity. 

The Summary

The research has resulted in a series of recommendations for both industry and for future study. 

Included is for daily recutting of stems to inhibit the full effects of a wounding response. 

While this will trigger a new wounding reaction, it possibly will delay the effects of suberin formation and impaired water uptake. 

The work also shows improved vase life can be achieved by standing the stems in deep water in the packing shed before despatch. 

And cut flower growers/exporters/consumers can apply a short-term pulse of S-carvone as a new, safe and easy-to-use suberin inhibitor. 

Finally, as a by-product of the work, several Australian flowers of commercial and export importance are now known to be sensitive to ethylene and therefore should be treated and/or protected against its effects in order to attain optimum vase life. These are Baeckea virgata, and Crowea exalata. A New Zealand native Lophomyrtus x ralphii’ Krinkly’. 

Acacia floribunda was previously shown to be ethylene insensitive. 

The report says the ethylene sensitivity of the following flowers and foliage either was not tested (nt) or its sensitivity remains unclear (Acacia Baileyana (nt &u); Baeckea behrii (nt &u); Ceratopetalum gummiferum NSW Christmas Bush/Festival Bush (nt &u); Grevillea longistyla (nt but probably not sensitive) Hakea terestifolia (nt); Hakea francisiana (nt) Leptospermum obovatum (nt); Leptospermum polygalifolium (nt) ;Rosa hybrida’ Lambada (ut); and Spaerolobium vimineum (nt) . 

The researchers report that the project has begun a new area of cut flower research. This has involved examining cut flower stems under Transmission Electron Microscopy. It also has confirmed the often-hypothesised anatomical nature of the physiological occlusion that occurs in cut flowers. 

They say more work clearly has to be done in this area including a daily time-course of the wounding reponse in H. francisiana to see whether suberin does eventually form. There also needs to be examination of numerous other species, especially short-lived and long-living cut flowers. 
 

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